Chronic Exposure to Wildland Fire Smoke Exposure Impairs Vascular Function in Hypercholesterolemic Mice

Introduction:: Historical data from the U.S. Environmental Protection Agency reveal that wildland fire smoke (WFS) contributes to nearly one third of the respirable particulate matter (PM) and carbon monoxide (CO) in the air [1]. Epidemiological evidence shows that inhalation of PM from WFS escalates the immediate risk of all-cause and cardiovascular (CV) mortality [2]. It is reasonable to expect that repeated insults over time may cause permanent damage to CV tissues and organs, particularly in wildland firefighters (WLFFs) who experience high-intensity exposure because of their proximity to the fire during active duty and at basecamp. Although a significant positive correlation between years of firefighting service and likelihood of developing hypertension and arrhythmia has been established [3], the occupational CV hazards of WFS inhalation remain unclear. To complement the sparse epidemiological data, we inferred the CV outcomes of chronic WFS inhalation by exposing mice to lab-scale WFS, for time and concentration conditions equivalent to an average mid-career WLFF [4]. Our findings provide scientific evidence supporting the development of respiratory protective standards and preventative measures to protect WLFF health.

Materials and Methods:: Lab-scale smoldering Douglas fir smoke (DFS) at target PM concentration of 40 mg/m³ was generated using a custom-built apparatus [4]. Male, 8-week-old Apoe^-/- mice were exposed to either DFS (N=20) or filtered room air (N=20) for 2 hrs/day, 5 days/week, over 2 or 4 months. DFS emissions were assessed daily for PM (gravimetrically) and CO (Enerac 700). Blood pressure was monitored weekly (CODA, Kent Scientific) and carboxyhemoglobin (COHb) levels were checked after the first day of exposure and at both endpoints (ABL80 FLEX, Radiometer). Blood serum was collected longitudinally over the course of the 4-month exposure for cytokine analysis (Q-Plex, Quansys Biosciences). Mice were sacrificed after 2 and 4 months of exposure to harvest the abdominal aorta for in vitro passive and vasoactive mechanical testing. Aortic samples were first pre-constricted with phenylephrine to examine endothelium-dependent vasodilatation (EDV) via luminal administration of acetylcholine at increasing concentrations (10^-9 to 10^-4 M). The contribution of eNOS-derived nitric oxide (NO) to EDV was then evaluated following the same protocol but after preincubation with the eNOS blocker NG-Nitroarginine methyl ester hydrochloride (L-NAME). Constitutive parameters of aortic tissues were estimated from cyclic inflation-extension tests and used to predict passive distensibility under in-vivo loads. Immunofluorescence was performed on aortic tissues to quantify microstructural composition and probe endothelial signaling cascades.

Results, Conclusions, and Discussions:: Average PM and CO concentrations in DFS over the 4 months of exposure reached 39±13 mg/m³ and 218±46 ppm, respectively, and yielded average COHb levels of 21.2±4.8 %. No PM or CO were detected in filtered air and control mice exhibited average COHb concentration of 1.7±0.9 %. Systolic blood pressure raised over the first 3 months of DFS exposure and stabilized thereafter, while it remained at baseline in controls (Air: 99±1, DFS_2mo: 105±1, DFS_4mo: 107±1 mmHg). This small yet significant change in hemodynamic loads was associated with progressive wall thickening (Air: 38±1, DFS_2mo: 40±1, DFS_4mo: 44±1 mm) and loss of distensibility (Air: 28±0.7, DFS_2mo: 25±0.7, DFS_4mo: 22±0.7 MPa^-1) in the aorta. Increased collagen I content in the media augmented wall thickness at both endpoints, while collagen I and III deposition in the adventitia was detected after 4 months of DFS exposure. Comparison of the response to acetylcholine with or without preincubation with L-NAME showed significant contribution of eNOS-produced NO to the EDV in the control aorta, but revealed a deficit in the DFS aorta at both endpoints. DHE staining showed elevated superoxide levels in the aorta after 2 and 4 months of DFS exposure, compared to their respective controls. Preincubation with L-NAME reduced superoxide staining, suggesting that uncoupled eNOS supplemented reactive oxygen species (ROS) in the DFS groups. Decline in DHE fluorescence upon pre-incubation with the NOX2 inhibitor GSK, as well as the overexpression of the NOX2 protein, indicated that activation of NOX2 in endothelial cells augmented ROS levels and contributed to eNOS uncoupling in aortic samples from mice exposed to DFS. Circulating TNF-a showed a biphasic response with peaks at 7 days and 3 months of DFS exposure and expression of TNF-a in the endothelium increased at both endpoints, suggesting that inflammation plays a role in WFS-induced cardiovascular damage.
Collectively, our findings show that DFS exposure increases the inflammatory and oxidative burden in the aorta, leading to eNOS uncoupling and collagen driven remodeling. Impairment of the vasoactive function, alongside thickening and stiffening the wall, may elevate blood pressure, thereby increasing the workload on the heart and damaging the microvasculature.


Acknowledgements (Optional): : This work was supported by DHS/FEMA (EMW-2017-FP-00446) and NIH/NIEHS (R01E5033792).

References (Optional): : [1] US Environmental Protection Agency, Air emissions inventories. criteria pollutants national tier 1 for 1970 - 2020. Available at https://www.epa.gov/air-emissions-inventories.

[2] A. Doubleday, J. Schulte, L. Sheppard, M. Kadlec, R. Dhammapala, J. Fox, T. B. Isaksen (2020) Mortality associated with wildfire smoke exposure in Washington state, 2006–2017: a case-crossover study (2020). Environmental Health 19(1): 1–10.

[3] E. O. Semmens, J. Domitrovich, K. Conway, C. W. Noonan (2016) A cross-sectional survey of occupational history as a wildland firefighter and health. American Journal of Industrial Medicine 59(4): 330–335.

[4] Eden MJ, Matz J, Garg P, Gonzalez MP, McElderry K, Wang S, Gollner MJ, Oakes JM, Bellini C (2023). Prolonged smoldering Douglas fir smoke inhalation augments respiratory resistances, stiffens the aorta, and curbs ejection fraction in hypercholesterolemic mice. Science of the Total Environment 861:160609.